PROJECT SUMMARY Nociception is the process by which sensory neurons detect painful stimuli. Nociceptor activation can initiate both an acute pain response and produce local inflammation leading to tissue acidosis. This reduction in pH is associated with a range of pathophysiological responses in the orofacial region including pericoronitis, pulpitis and migraine. Acid sensing ion channels (ASICs) are molecular proton sensors that are activated in response to this extracellular acidification. ASICs are widely expressed throughout both the peripheral and central nervous systems (PNS/CNS). In the PNS, ASICs are found in the trigeminal ganglion (TG) neurons that innervate tooth pulp, facial skin and periodontal Ruffini endings. There is evidence that ASIC3 expression correlates with orofacial pain following experimental tooth pain in rats, which could be relieved with ASIC antagonists, APETx2 and amiloride. ASICs involvement in nociception and mechanosensation within TG neurons make it a novel analgesic target, but much remains to be elucidated about ASICs molecular mechanisms before robust and effective drugs can be developed. While structures of ASIC have been solved, revealing in atomic detail the trimeric nature of this channel, they lack the intracellular termini. Using novel fluorescence methodologies like specific labeling with an unnatural amino acid and transition metal ion FRET accompanied by electrophysiology, specific aim 1 seeks to examine intramolecular dynamic rearrangements of the intracellular domains during channel activation. This set of experiments will fill a void by answering questions that the ASIC structures do not. I will seek to determine the dynamic rearrangements of the n- terminus during channel function. I will assign these rearrangements in channel structure to functional states of the channel using patch clamp electrophysiology. In addition, I will test some hypotheses, put forth by the crystal structures. There is an unusual domain swapped architecture in the transmembrane domains of ASIC in some, but not all, crystal structures. I can use my novel approach to test for the presence of this swap in real membranes. In aim 2, I will seek to understand the heteromeric assembly of ASICs. The literature overwhelmingly focuses on homomers likely due to the ease of studying one subunit at a time. The physiological relevance of ASIC heteromers makes them critical to study. However, results from heteromeric studies are challenging to interpret, often because channel stoichiometry is unknown. Specific aim 2 will try to delineate the rules of ASIC heteromerization. These experiments will begin to answer questions which should motivate future studies of heteromers in addition to homomers. Do heteromers form preferentially, whether that be 2:1 or 1:2, or is it nonspecific? What sites on the channel are responsible for heteromerization? These findings will help elucidate the molecular mechanisms of ASIC gating and provide a new understanding on the heteromeric assembly of these channels. My goal is to ultimately inform drug design targeting ASIC as a treatment for inflammatory orofacial pain.